D-Glucosamine Assay Kit

As one of the most frequently occurring monomers in the biosphere, glucosamine is a valuable metabolite for several applications. Although microbial glucosamine production is still in its infancy, it offers the possibility to circumvent problems associated with traditional production by hydrolysis. Of particular interest is a study with Aspergillus niger, which reports for the first time high glucosamine excretion in the early phase of citric acid production. These results have relevance for both the commercial glucosamine production and deeper insight into the regulation of organic acid excretion in fungi. To investigate glucosamine excretion, we performed bioreactor batch cultivations with Penicillium ochrochloron CBS123.824 and A. niger CBS120.49 using cultivation conditions which are known to trigger the production of citric acid. Glucosamine detection in culture filtrates was achieved by two photometric methods, High performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) and HPLC with mass spectrometry detection (HPLC-MS). Surprisingly, we detected no glucosamine at all. Based on a critical review of published data for A. niger, we conclude that the reported high levels of excreted glucosamine might be an experimental artifact. However, growth experiments with glucosamine as a combined or single source for carbon or nitrogen showed that both organisms are in principle able to transport glucosamine across their plasma membrane, which is a prerequisite for the excretion of glucosamine.

The hexosamine biosynthetic pathway (HBP) plays essential roles in growth and development in plants. However, insight into the biological function of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), mediating the first regulatory step of the HBP, remains unclear in plants. Here, we report the molecular characterization of Arabidopsis AtGFAT1 gene. AtGFAT1 was highly expressed in mature pollen grains, but its expression was not detectable in the rest of the organs. Pollen grains bearing the gfat1-2 knockout allele displayed defects in a polar deposition of pectin and callose in the pollen cell wall, leading to no genetic transmission of the gfat1-2 allele through the male gametophyte. AtGFAT1 overexpression increased glucosamine (GlcN) content and enhanced resistance to tunicamycin (Tm) treatment, while RNAi-mediated suppression reduced GlcN content and resistance to Tm treatment. However, the decrease in Tm resistance by RNAi suppression of AtGFAT1 was recovered by a GlcN supplement. The exogenous GlcN supplement also rescued gfat1-2/gaft1-2 mutant plants, which were otherwise not viable. The gfat1-2/gfat1-2 plants stopped growing at the germination stage on GlcN-free medium, but GlcN supplement allowed wild-type growth of gfat1-2/gfat1-2 plants. In addition, reactive oxygen species production, cell death and a decrease in protein N-glycosylation were observed in gfat1-2/gaft1-2 mutant plants grown on GlcN-free medium, whereas these aberrant defects were not detectable on GlcN-sufficient medium. Taken together, these results show that the reduction of protein N-glycosylation was at least partially responsible for many aberrant phenotypes in growth and development as well as the response to Tm treatment caused by AtGFAT1 deficiency in Arabidopsis.

Entomopathogenic fungi infect insects via penetration through the cuticle, which varies remarkably in chemical composition across species and life stages. Fungal infection involves the production of enzymes that hydrolyse cuticular proteins, chitin and lipids. Host specificity is associated with fungus–cuticle interactions related to substrate utilization and resistance to host-specific inhibitors. The soil fungus Conidiobolus coronatus (Constantin) (Entomophthorales: Ancylistaceae) shows virulence against susceptible species. The larvae and pupae of Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae), Calliphora vomitoria (Linnaeus), Lucilia sericata (Meigen) (Diptera: Calliphoridae) and Musca domestica (Linnaeus) (Diptera: Muscidae) are resistant, but adults exposed to C. coronatus quickly perish. Fungus was cultivated for 3 weeks in a minimal medium. Cell-free filtrate, for which activity of elastase, N-acetylglucosaminidase, chitobiosidase and lipase was determined, was used for in vitro hydrolysis of the cuticle from larvae, puparia and adults. Amounts of amino acids, N-glucosamine and fatty acids released were measured after 8 h of incubation. The effectiveness of fungal enzymes was correlated with concentrations of compounds detected in the cuticles of tested insects. Positive correlations suggest compounds used by the fungus as nutrients, whereas negative correlations may indicate compounds responsible for insect resistance. Adult deaths result from the ingestion of conidia or fungal excretions.

Chicken cartilage (claw) is a waste of chicken cuts which are widely available in Indonesia. Cartilage part of chicken claw becomes a potential source of chondroitin sulfate (CS) and glucosamine (GS). This study aims to determine the most optimal extraction methods of CS and GS from cartilage of chicken claw. Various types of extraction methods used in this study are taken from the extraction by using boiling water (2 and 2.5 hours), acetic acid (7 and 17 hours), as well as proteolysis by papain (24 and 48 hours). Parameters observed include chemical characteristics of powdered cartilage of chicken claw as well as CS and GS levels in powdered cartilage of chicken claw extract. The results of this research show that the levels of CS and GS of chicken claw cartilage powder were 2.17% and 13%. Meanwhile, the highest GS level was obtained from the extraction with water treatment for 2.5 hours which was 8.1%. The treatment and duration of extraction will significantly affect the number of GS which was produced. The highest content of CS was obtained from the extraction with the enzyme treatment for 48 hours which was 2.47%. The best treatment is the extraction with water treatment for 2.5 hours which were the extracts with GS levels of 8.1% and 2.03% CS was selected through the analysis of multiple attribute.

Embryo implantation in the uterus depends on decidualization of the endometrial stromal cells (ESCs), and glucose utilization via the pentose phosphate pathway is critical in this process. We hypothesized that the amino sugar glucosamine may block the pentose phosphate pathway via inhibition of the rate-limiting enzyme glucose-6-phosphate dehydrogenase in ESCs and therefore impair decidualization and embryo implantation, thus preventing pregnancy. Both human primary and immortalized ESCs were decidualized in vitro in the presence of 0, 2.5, or 5 mM glucosamine for 9 days. Viability assays demonstrated that glucosamine was well tolerated by human ESCs. Exposure of human ESCs to glucosamine resulted in significant decreases in the activity and expression of glucose-6-phosphate dehydrogenase and in the mRNA expression of the decidual markers prolactin, somatostatin, interleukin-15, and left-right determination factor 2. In mouse ESCs, expression of the decidual marker Prp decreased upon addition of glucosamine. In comparison with control mice, glucosamine-treated mice showed weak artificial deciduoma formation along the stimulated uterine horn. In a complementary in vivo experiment, a 60-day-release glucosamine (15, 150, or 1500 µg) or placebo pellet was implanted in a single uterine horn of mice. Mice with a glucosamine pellet delivered fewer live pups per litter than those with a control pellet, and pup number returned to normal after the end of the pellet-active period. In conclusion, glucosamine is a nonhormonal inhibitor of decidualization of both human and mouse ESCs and of pregnancy in mice. Our data indicate the potential for development of glucosamine as a novel, reversible, nonhormonal contraceptive.